Project Summary In the US, stroke is the third leading cause of death and primary cause of severe disability, with over 700,000 individuals experiencing an ischemic episode each year. Tissue plasminogen activator, the only FDA approved thrombolytic drug for stroke, must be infused within a short period following the initial ischemia and has limited benefits and outcomes. Stroke therapy using adult stem cells such as human mesenchymal stem cells (hMSC) from bone marrow and adipose tissue sources has emerged as a promising avenue to reduce and reverse neurodegeneration resulting from stroke by promoting tissue protection and repair. The project seeks to (a) investigate the in vivo fate and mechanisms of implanted hMSC that underlie their homing and therapeutic benefits in stroke treatment using high field magnetic resonance imaging (MRI) and spectroscopy (MRS) and (b) define expansion and pre-conditioning culture environments that preserve hMSC therapeutic potency. The project's hypothesis is that hMSC expansion conditions impact efficacy in stroke treatment though hypoxic induction factors, and these impacts can be monitored and further optimized with time course information gathered by in vivo high field MRI and MRS about functional recovery. The project is built upon complementary expertise and preliminary results in high field MRI analysis of hMSC fate in stroked animals as well as bioreactor systems for hMSC expansion. The project will investigate the migration and engraftment of culture-expanded hMSC within stroke lesions by MR techniques and delineate the impact of hypoxic pre- conditioning and aggregation on hMSC in vivo fate. The project also will evaluate the interactions of hMSC and endogenous neuroprogenitors and their synergist role in stroke lesion recovery. The goal of these efforts will be to determine achieve timed injections of culture expanded hMSC during stroke recovery and determine the efficacy of hMSC therapy beyond the current window of thrombolytic drugs. Additionally, as a means of translating findings, the project investigates bioreactor conditions that enable scalable expansion to increase availability to human patients and impact therapeutic efficacy. The success of this project will expand knowledge about in vivo hMSC fate as influenced by pre-activation, identify the mechanisms and range of impacts for hMSC action in stroke recovery, and establish a scalable bioreactor strategy for clinical translation. Relevance Current thrombolytic agents must be employed quickly after ischemic onset, limiting their effectiveness in all but 5% of stroke patients. hMSC promote stroke lesion recovery outside this acute window, but their eventual clinical application requires in-depth knowledge of mechanisms and biodistribution as well as the availability of a transplantable cell population. Utilizing a unique set of experiments, the project is designed to provide mechanistic insights into the efficacy of bioreactor expanded and pre-conditioned hMSC for stroke treatment.